The following disclosure through page 31 relates to a first embodiment of the present invention.
The first embodiment of the present invention relates to a sustained-release preparation which comprises a water-insoluble or a slightly water-soluble polyvalent metal salt of a water-soluble peptide type of physiologically active substance which is not an endothelin antagonist, and a biodegradable polymer.
Physiologically active substances, particularly peptides or derivatives thereof, are known to exhibit various pharmacologic actions in vivo. Some have been produced in large amounts, for pharmaceutical application, by chemical synthesis, or as a result of advances in gene engineering and cell engineering technologies, using organisms such as Escherichia coli, yeasts, animal cells and hamsters. However, these peptides must be administered frequently, since they generally have a short biological half-life, and so pose a significant physical burden of injection on patents. To solve this problem, various attempts have been made to develop sustained-release preparations.
The first problem to solve in developing a sustained-release preparation of a water-soluble physiologically active substance, particularly a water-soluble peptide (hereinafter also referred to as xe2x80x9cpeptidexe2x80x9d) is to control peptide solubility, i.e., to regulate the peptide release rate.
Japanese Publication of the Translation of International Patent Application No. 500286/1991 discloses an insoluble zinc-protamine-xcex1-interferon complex.
Japanese Patent Unexamined Publication No. 2930/1988 discloses a system comprising a polylactide in which a macromolecular polypeptide is dispersed.
Japanese Patent Unexamined Publication Nos. 221855/1993 and 172208/1994 disclose a technology by which a water-soluble peptide is converted to a water-insoluble peptide salt, which is then suspended in an organic medium containing a biodegradable polymer to efficiently incorporate the water-soluble peptide in fine grains. The water-insoluble peptide used in these patent publications is an organic acid salt formed at the base portion of the water-soluble peptide molecule, and is exemplified by pamoate, tannic acid, stearic acid or palmitate.
Although there have been various attempts to produce sustained-release preparations of water-soluble physiologically active substances, as stated above, no satisfactory sustained-release preparations have been obtained; there is therefore need for the development of a sustained-release preparation that is highly efficient in incorporating water-soluble physiologically active substance, suppresses initial water-soluble physiologically active substance burst, offers a constant water-soluble physiologically active substance release rate, and keeps the bioactivity of water-soluble physiologically active substance.
Through extensive investigation to solve the above problems, the present inventors found that a sustained-release preparation, having dramatically increased efficiency of water-soluble peptide type of physiologically active substance except for an endothelin antagonist incorporation in a biodegradable polymer and showing little drug burst just after administration to the living body, can be obtained by producing a water-insoluble or a slightly water-soluble polyvalent metal salt of a water-soluble peptide type of physiologically active substance except for an endothelin antagonist (hereinafter also referred to as xe2x80x9ccomplexxe2x80x9d), which salt is formed from a combination of a water-soluble peptide type of physiologically active substance except for an endothelin antagonist having an acidic group, or a water-soluble salt thereof (hereinafter also referred to as xe2x80x9cphysiologically active substancexe2x80x9d), with a water-soluble polyvalent metal salt, and dispersing or dissolving it in a biodegradable polymer. After further investigations based on this finding, the inventors developed the present invention.
Accordingly, the present invention relates to:
(1) a sustained-release preparation which comprises
(a) a water-insoluble or slightly water-soluble polyvalent metal salt of a water-soluble peptide type of physiologically active substance except for an endothelin antagonist and
(b) a biodegradable polymer,
(2) a preparation of term 1 above, wherein the physiologically active substance is a water-soluble peptide or a derivative thereof,
(3) a preparation of term 2 above, wherein the peptide is a hormone, cytokine, hematopoietic factor, growth factor, enzyme, soluble or solubilized receptor, antibody, antigen containing peptide, blood coagulation factor or adhesion molecule,
(4) a preparation of term 2 above, wherein the peptide is a hormone,
(5) a preparation of term 4 above, wherein the hormone is a growth hormone
(6) a preparation of term 3 above, wherein the hormone is an insulin,
(7) a preparation of term 2 above, wherein the peptide is a cytokine,
(8) a preparation of term 7 above, wherein the cytokine is an interferon,
(9) a preparation of term 2 above, wherein the peptide is a growth factor,
(10) a preparation of term 1 above, wherein the polyvalent metal salt is a transition metal salt,
(11) a preparation of term I above, wherein the polyvalent metal salt is a zinc salt,
(12) a preparation of term 1 above, wherein the solubility of the polyvalent metal salt to water is about 0 to about 0.1% (w/w) at 20xc2x0 C.,
(13) a preparation of term 1 above, wherein the solubility of the polyvalent metal salt to water is about 0 to about 0.01% (w/w),
(14) a preparation of term 1 above, which contains about 0.1 to about 50% (w/w) of the polyvalent metal salt,
(15) a preparation of term 1 above, which contains about 1 to about 30% (w/w) of the polyvalent metal salt,
(16) a preparation of term 1 above, wherein the biodegradable polymer is an aliphatic polyester,
(17) a preparation of term 16 above, wherein the aliphatic polyester is a polymer of lactic acid and glycolic acid,
(18) a preparation of term 17 above, wherein the composition ratio of lactic acid and glycolic acid is 100/0 to about 40/60 (mole %),
(19) a preparation of term 18 above, wherein the composition ratio is about 90/10 to about 45/55 (mole %),
(20) a preparation of term 17 above, wherein the weight-average molecular weight of the polymer is about 3,000 to about 20,000,
(21) a preparation of term 17 above, wherein the weight-average molecular weight of the polymer is about 3,000 to about 14,000,
(22) a preparation of term 16 above, wherein the alihatic polyester is a homopolymer of lactic acid,
(23) a preparation of term 22 above, wherein the weight-average molecular weight of the homopolymer is about 3,000 to about 20,000,
(24) a preparation of term 22 above, wherein a weight-average molecular weight of the homopolymer is about 3,000 to about 14,000,
(25) a preparation of term 1 above, wherein the preparation is a microcapsule,
(26) a preparation of term 25 above, wherein the microcapsule is for injection,
(27) a preparation of term 1 above, which is an injectable one,
(28) Use of a water-insoluble or slightly water-soluble polyvalent metal salt of a water-soluble peptide type of physiologically active substance except for an endothelin antagonist and a biodegradable polymer for the production of a sustained-release preparation, and
(29) a method of producing a sustained-release preparation, which comprises dispersing a water-insoluble or slightly water-soluble polyvalent metal salt of a water-soluble peptide type of physiologically active substance except for an endothelin antagonist in an oil phase containing a biodegradable polymer to make a solid-in-oil emulsion, adding the solid-in-oil emulsion to a water phase to make a solid-in-oil-in-water emulsion, and then in-water drying the soild-in-oil-in-water emulsion.
Incidentally abbreviations of amino acid, peptide or the like used in the present invention are based on those in accordance with IUPAC-IUB Commission on Biochemical Nomenclature or those conventionally used in the relevant fields, and possible optical isomers of amino acid are, unless otherwise specified, L-isomers.
The physiologically active substance in the water-insoluble or the slightly water-soluble polyvalent metal salt is a physiologically active substance having an acidic group. Here, the acidic group is exemplified by the carboxyl group and sulfo group. The physiologically active substance is preferably a physiologically active substance having a peptide bond or an amino acid and acidic group.
The acidic group may be derived from an amino acid. More preferably, the physiologically active substance is a water-soluble peptide having an acidic group or a derivative thereof. A solubility of the physiologically active substance to water is 1% (w/w) or more at 25xc2x0 C.
The physiologically active substance preferably has two or more carboxyl groups.
The molecular weight of the physiologically active substance is about 200 to 200,000, preferably about 200 to about 50,000, more preferably about 500 to about 40,000.
A representative activity of a physiologically active substance is hormone action. The physiologically active substance may be a natural, synthetic, semi-synthetic or genetically engineered product, or a derivative thereof. As concerns the mechanism of action, these physiologically active substances may be agonistic or antagonistic.
Physiologically active substances, particularly water-soluble peptide or a derivative thereof for the present invention include hormones, cytokines, hematopoietic factors, growth factors, enzymes, a soluble or solubilized receptor, an antibody or a fragment thereof, an antigen containing peptide, a blood coagulation factor, an adhesion molecule, agonists or antagonists capable of binding to receptors of the physiologically active substances and so on.
Example hormones include insulin, growth hormone, natriuretic peptide, gastrin, prolactin, adrenocortico-tropic hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), motilin, kallikrein and so on. The hormone is preferably insulin and growth hormone.
Example cytokines include lymphokines, monokines and so on. Example lymphokines include interferons (alpha, beta, gamma), interleukins (IL-2 through IL-12) and so on. Example monokines include an interleukin 1 (IL-1), tumor necrosis factor and so on. The cytokine is preferably a lymphokine, more preferably an interferon (alpha, beta, gamma).
Example hematopoietic factors include erythropoietin, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), thrombopoietin, platelet growth-stimulating factor, megakaryocyte potentiator and so on.
Example growth factors include basic or acidic fibroblast growth factors (FGF), members of the family thereof (e.g., FGF-9 etc.), nerve cell growth factor (NGF) or members of the family thereof, insulin-like growth factors (e.g., IGF-1, IGF-2), bone morphogenetic protein (BMP) or members of the family thereof and so on.
Example enzymes include superoxide dismutase (SOD), tissue plasminogen activator (TPA) and so on.
Example soluble receptors include soluble IL-6 receptor, insulin-like growth factor binding protein (IGFBP), soluble TNF receptor, soluble EGF receptor, soluble IL-1 receptor and so on.
Example solubilized receptors include a known receptors such as IL-1 receptor, IL-6 receptor, TNF receptor or Fas ligand etc., which is solubilized by a method of gene engineering.
Example antibodies include a human monoclonal antibody, a human-mouse chimeric monoclonal antibody in which the variable region of an antibody derived from mouse is bound to the constant region of an antibody derived from human, or a fragment thereof and so on. Example type of antibody include IgM, IgG, IgE and so on. Example antigenes, which is recognized by the above described antibody, include platelet, virus and so on.
Example blood coagulation factors include factor VIII and so on.
Example adhesion molecules include fibronectin, ICAM-1 and so on.
Furthermore, example physiologically active substances include endothelin, Arg-Gly-Asp-Ser (RGDS), pituitary adenylate cyclase activating polypeptide (PACAP) and so on.
The physiologically active substance is converted to a water-insoluble or a slightly water-soluble polyvalent metal salt thereof by bringing it into contact with a water-soluble polyvalent metal SAH.
The polyvalent metal in the water-soluble polyvalent metal salt is exemplified by divalent, trivalent or tetravalent metal etc. such as alkaline earth metals (e.g., calcium, magnesium etc.), transition metals [e.g., iron (II, III), copper (II), zinc (II) etc.), the group IIIb metals [e.g., aluminum (II, III) etc.], the group IVb metals [e.g., tin (II, IV) etc.] and so on. The polyvalent metal is preferably alkaline earth metals or transition metals, more preferably calcium or zinc, still more preferably zinc.
Water-soluble polyvalent metal salts include salts of polyvalent metals and acids, e.g., salts of polyvalent metals and inorganic acids, and salts of polyvalent metals and organic acids.
The salt of a polyvalent metal and an acid is preferably a salt whose water solubility at normal temperature (20xc2x0 C.) is not lower than about 20 mg/ml, more preferably not lower than about 100 mg/ml, and still more preferably not lower than about 200 mg/ml.
Inorganic acids to form salts with polyvalent metals include hydrochloric acid, sulfuric acid, nitric acid, thiocyanic acid and so on.
Organic acids to form salts with polyvalent metals include aliphatic carboxylic acids and aromatic acids. The aliphatic carboxylic acid is preferably an aliphatic carboxylic acid having 2 to 9 carbon atoms. Aliphatic carboxylic acids include aliphatic monocarboxylic acids, aliphatic dicarboxylic acids, aliphatic tricarboxylic acids and so on. These aliphatic carboxylic acids may be saturated or unsaturated one.
Example aliphatic monocarboxylic acids include saturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (e.g., acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, caprynic acid etc.) and unsaturated aliphatic monocarboxylic acids having 2 to 9 carbon atoms (e.g., acrylic acid, propiolic acid, methacrylic acid, crotonic acid, isocrotonic acid etc.).
Example aliphatic dicarboxylic acids include saturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (e.g., malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid etc.) and unsaturated aliphatic dicarboxylic acids having 2 to 9 carbon atoms (e.g., maleic acid, fumaric acid, citraconic acid, mesaconic acid etc.).
Example aliphatic tricarboxylic acids include saturated aliphatic tricarboxylic acids having 2 to 9 carbon atoms (e.g., tricarballylic acid, 1,2,3-butanetricarboxylic acid etc.).
The above-mentioned aliphatic carboxylic acids may have 1 or 2 hydroxyl groups. Such aliphatic carboxylic acids include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, citric acid and so on.
The aliphatic carboxylic acid is preferably an aliphatic monocarboxylic acid, more preferably an aliphatic monocarboxylic acid having 2 to 9 carbon atoms, and still more preferably a saturated aliphatic monocarboxylic acid having 2 or 3 carbon atoms. Examples of particularly preferable aliphatic carboxylic acids include acetic acid and so on.
Example aromatic acids include benzoic acid, salicylic acid and so on, with preference given to benzoic acid.
Examples of salts of polyvalent metals and inorganic acids, i.e., inorganic acid polyvalent metal salts, include halides (e.g., zinc chloride, calcium chloride), sulfates, nitrates, thiocyanates and so on.
Examples of salts of polyvalent metals and aliphatic carboxylic acids, i.e., aliphatic carboxylic acid polyvalent metal salts, include calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, zinc tartrate and so on. Preferable aliphatic carboxylic acid polyvalent metal salts include calcium acetate and zinc acetate. Greater preference is given to zinc acetate.
Examples of salts of polyvalent metals and aromatic acids, i.e., aromatic acid polyvalent metal salts, include benzoates, salicylates and so on. Greater preference is given to zinc benzoate.
A water-insoluble or a slightly water-soluble polyvalent metal salt of a physiologically active substance is produced by mixing in a solvent the water-soluble physiologically active substance and a water-soluble polyvalent metal salt. The mixing procedure is preferably conducted in water.
The mixing ratio (mole ratio) of the physiologically active substance and water-soluble polyvalent metal salt in water is, for example 1:1 to 1:1000, preferably 1:1 to 1:100, more preferably 1:1 to 1:50, still more preferably 1:1 to 1:10. The concentrations of both components in water may be optional, as long as they exceed the solubility of the resulting complex, within their respective solubility ranges.
The pH of the aqueous solution resulting from the above mixing must be such that the bioactivity of the physiologically active substance is not affected, and that the solubilities of the physiologically active substance and water-soluble polyvalent metal salt are not lowered in excess. Although the mixing procedure is normally conducted in distilled water, it may be conducted in water adjusted to weakly acidic, neutral, or weakly alkaline pH as necessary.
xe2x80x9cBeing water insoluble or slightly water-solublexe2x80x9d as mentioned herein is not irreversible but reversible, meaning that water solubility is very low. Water solubility is about 0 to about 0.1% (w/w), preferably about 0 to about 0.01% (w/w) at ordinary temperature (20xc2x0 C.).
The thus-obtained water insoluble or slightly water-soluble polyvalent metal salt of a water-soluble physiologically active substance is used after being vacuum dried or freeze dried as necessary.
In the sustained-release preparation of the present invention, the content of the water-insoluble or slightly water-soluble polyvalent metal salt of the physiologically active substance is normally about 0.1 to about 50% (w/w), preferably about 1 to about 30% (w/w).
The biodegradable polymer is exemplified by high-molecular polymers slightly soluble or insoluble in water, such as aliphatic polyesters (e.g., homopolymers, copolymers or mixtures thereof synthesized from one or more xcex1-hydroxycarboxylic acids such as glycolic acid, lactic acid, hydroxybutyric acid etc.), hydroxydicarboxylic acids such as malic acid etc., hydroxytricarboxylic acids such as citric acid etc. and others, poly-xcex1-cyanoacrylic acid esters, polyamino acids such as poly-xcex3-benzyl-L-glutamic acid and so on. These may be used in mixture at appropriate ratios. The type of polymerization may be random, block or graft.
The biodegradable polymer is preferably an aliphatic polyester (e.g., a homopolymer, copolymer or mixture thereof synthesized from one or more xcex1-hydroxycarboxylic acids such as glycolic acid, lactic acid, hydroxybutyric acid etc., hydroxydicarboxylic acids such as malic acid etc., hydroxytricarboxylic acids such as citric acid etc. and others).
Of the above-mentioned aliphatic polyesters, homopolymers or copolymers synthesized from one or more xcex1-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid etc.) are preferred from the viewpoint of reliable biodegradability and biocompatibility. More preferably, the aliphatic polyester is a copolymer synthesized from one or more xcex1-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid etc.). Also, these copolymers may be used in mixture.
The biodegradable polymer for the present invention is produced by a commonly known method.
Although the above-described xcex1-hydroxycarboxylic acid may be of the D-, L- or D,L-configuration, it is preferable that the ratio of the D-/L-configuration (mole %) fall within the range from about 75/25 to about 25/75. The ratio of the D-/L-configuration (mole %) is more preferably about 60/40 to about 30/70.
Example copolymers of the above-described xcex1-hydroxycarboxylic acid include copolymers of glycolic acid with another xcex1-hydroxy acid, which is preferably lactic acid or 2-hydroxybutyric acid.
The xcex1-hydroxycarboxylic acid copolymer is preferably a lactic acid-glycolic acid copolymer or a 2-hydroxybutyric acid-glycolic acid copolymer.
More preferably, the xcex1-hydroxycarboxylic acid copolymer is a lactic acid-glycolic acid copolymer.
With respect to the lactic acid-glycolic acid copolymer, it is preferable that the content ratio (lactic acid/glycolic acid) (mole %) be about 100/0 to about 40/60. The content ratio is more preferably about 90/10 to about 45/55, and more preferably about 80/20 to about 45/55. The weight-average molecular weight of the lactic acid-glycolic acid copolymer is about 3,000 to about 20,000, preferably about 3,000 to about 14,000 more preferably about 3,000 to about 12,000.
Also, the degree of dispersion of the lactic acid-glycolic acid copolymer (weight-average molecular weight/number-average molecular weight) is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5.
The lactic acid-glycolic acid copolymer can be synthesized by a known process, such as the method described in Japanese Patent Unexamined Publication No. 28521/1986. It is preferable that the copolymer be synthesized by catalyst-free dehydration polymerization condensation.
With respect to the 2-hydroxybutyric acid-glycolic acid copolymer, it is preferable that glycolic acid account for about 10 to about 75 mole % and 2-hydroxybutyric acid for the remaining portion. More preferably, glycolic acid accounts for about 20 to about 75 mole %, and still more preferably about 30 to about 70 mole %. The weight-average molecular weight of the 2-hydroxybutyric acid-glycolic acid copolymer is preferably about 2,000 to about 20,000. The degree of dispersion of the 2-hydroxybutyric acid-glycolic acid copolymer (weight-average molecular weight/number-average molecular weight) is preferably about 1.2 to 4.0, more preferably about 1.5 to 3.5. A 2-hydroxybutyric acid-glycolic acid copolymer can be synthesized by a known process, such as that described in Japanese Patent Unexamined Publication No. 28521/1986. It is preferable that the copolymer be synthesized by catalyst-free dehydration polymerization condensation.
Preferable example homopolymers of the above-described xcex1-hydroxycarboxylic acid include homopolymer of lactic acid. The weight-average molecular weight of the homopolymer of lactic acid is about 3,000 to about 20,000, preferably about 3,000 to about 14,000. A homopolymer of lactic acid can be synthesized by a known process, such as that described in Japanese Patent Unexamined Publication No. 28521/1986. It is preferable that the homopolymer be synthesized by catalyst-free dehydration polymerization condensation.
The above-described 2-hydroxybutyric acid-glycolic acid copolymer may be used in a mixture with polylactic acid. Although the polylactic acid may be of the D- or L-configuration or a mixture thereof, it is preferable that the ratio of the D-/L-configuration (mole %) fall within the range from about 75/25 to about 20/80. The ratio of the D-/L-configuration (mole %) is more preferably about 60/40 to about 25/75, and still more preferably about 55/45 to about 25/75. The weight-average molecular weight of polylactic acid is preferably about 1,500 to about 20,000, more preferably about 1,500 to 10,000. Also, the degree of dispersion of the polylactic acid is preferably about 1.2 to 4.0, more preferably about 1.5 to 3.5.
For producing polylactic acid, two methods are known: ring-opening polymerization of lactide, a dimer of lactic acid, and dehydration polymerization condensation of lactic acid. For obtaining a polylactic acid of relatively low molecular weight for the present invention, direct dehydration polymerization condensation of lactic acid is preferred. This method is, for example, described in Japanese Patent Unexamined Publication No. 28521/1986.
When a 2-hydroxybutyric acid-glycolic acid copolymer and polylactic acid are used in mixture, their mixing ratio is about 10/90 to about 90/10 (% by weight). The mixing ratio is preferably about 20/80 to 80/20, and more preferably about 30/70 to 70/30.
In the present specification, weight-average molecular weight is defined as the molecular weight obtained by gel permeation chromatography (GPC) with 9 polystyrenes as reference substances with respective weight-average molecular weights of 120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and 162. Number-average molecular weight based on GPC measurement is also calculated. The degree of dispersion is calculated from the weight-average molecular weight and the number-average molecular weight. Measurements were taken using a GPC column KF804Lxc3x972 (produced by Showa Denko) and an RI monitor L-3300 (produced by Hitachi, Ltd.) with chloroform as the mobile phase.
The above-described polymer and copolymer, synthesized by catalyst-free dehydration polymerization condensation, usually has a terminal carboxyl group.
In the present invention, the biodegradable polymer preferably has a terminal carboxyl group.
A biodegradable polymer having a terminal carboxyl group is a polymer in which the number-average molecular weight by GPC determination and that by terminal group determination almost agree.
By terminal group quantitation, number-average molecular weight is calculated as follows:
About 1 to 3 g of the biodegradable polymer is dissolved in a mixed solvent of acetone (25 ml) and methanol (5 ml), and the solution is quickly titrated with a 0.05 N alcoholic solution of potassium hydroxide while being stirred at room temperature with phenolphthalein as an indicator to determine the terminal carboxyl group content; the number-average molecular weight based on terminal group quantitation is calculated using the following equation:
Number-average molecular weight based on terminal group quantitation=20,000 A/B
A: Weight mass (g) of the biodegradable polymer
B: Amount (ml) of the 0.05 N alcoholic solution of potassium hydroxide added until the titration end point is reached
For example, in the case of a polymer having a terminal carboxyl group, and synthesized from one or more xcex1-hydroxy acids by catalyst-free dehydration polymerization condensation, the number-average molecular weight based on GPC measurement and the number-average molecular weight based on terminal group quantitation almost agree. On the other hand, in the case of a polymer having essentially no terminal carboxyl group, and synthesized from a cyclic dimer by ring-opening polymerization using a catalyst, the number-average molecular weight based on terminal group quantitation is significantly higher than the number-average molecular weight based on GPC determination. This difference makes it possible to clearly differentiate a polymer having a terminal carboxyl group from a polymer having no terminal carboxyl group.
While the number-average molecular weight based on terminal group quantitation is an absolute value, the number-average molecular weight based on GPC determination is a relative value that varies depending on various analytical conditions (e.g., kind of mobile phase, kind of column, reference substance, slice width chosen, baseline chosen etc.); it is therefore difficult to have an absolute numerical representation of the latter. However, the fact that the number-average molecular weight based on GPC determination and that based on terminal group quantitation almost agree means that the number-average molecular weight based on terminal group quantitation falls within the range from about 0.5 to about 2 times, preferably from about 0.8 to about 1.5 times, the number-average molecular weight based on GPC determination. Also, the fact that the number-average molecular weight based on terminal group quantitation is significantly higher than that based on GPC determination means that the number-average molecular weight based on terminal group quantitation is about 2 times or more the number-average molecular weight based on GPC determination.
The sustained-release preparation of the present invention is produced by dispersing in a biodegradable polymer a water-insoluble or a slightly water-soluble polyvalent metal salt of a physiologically active substance obtained by mixing the physiologically active substance and a water-soluble polyvalent metal salt. Methods of producing a sustained-release preparation include the in-water drying method, phase separation method, spray drying method, and modifications thereof.
Methods of producing a sustained-release preparation, e.g., microcapsules, are described below.
(i) In-water Drying Method (o/w method)
In this method, a solution of a biodegradable polymer in an organic solvent is first prepared. The organic solvent used to produce the sustained-release preparation of the present invention preferably has a boiling point not higher than 120xc2x0 C. Such organic solvents include halogenated hydrocarbons (e.g., dichloromethane, chloroform, carbon tetrachloride etc.), alcohols (e.g., ethanol, methanol), acetonitrile and so on. These may be used in mixture at appropriate ratios. For example, when a dichloromethane and alcohols are used in mixture, their mixing ratio (v/v) is about 1000/1 to about 1/1, preferably about 100/1 to about 1/1, still more preferably about 10/1 to about 2/1. The organic solvent is preferably dichloromethane and acetonitrile, and still more preferably dichloromethane. The concentration of the biodegradable polymer in the organic solvent solution is normally about 0.01 to about 80% (w/w), preferably about 0.1 to about 70% (w/w), and more preferably about 1 to about 60% (w/w), depending on the molecular weight of the biodegradable polymer, kind of organic solvent and so on.
To the organic solvent solution of the biodegradable polymer thus obtained, a water-insoluble or a slightly water-soluble polyvalent metal salt of a physiologically active substance is added or dissolved, after being freeze-dried or vacuum dried as necessary. In this operation, the amount of complex added is set so that the complex:biodegradable polymer weight ratio is up to about 1:2, preferably about 1:3.
The organic solvent solution thus prepared is added to an aqueous phase to fo,rm an o/w emulsion using a turbine type mechanical stirrer or the like, followed by evaporation of the solvent in the oil phase, to yield microcapsules. The volume of the aqueous phase is normally chosen over the range of about 1 to about 10,000 times, preferably about 2 to about 5,000 times, and more preferably about 5 to about 2,000 times, the volume of the oil phase.
An emulsifier may be added to the external aqueous phase. The emulsifier may be any one, as long as it is capable of forming a stable o/w emulsion. Examples of such emulsifiers include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, is polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid and so on. These may be used in combination as appropriate. The emulsifier concentration in the external aqueous phase is preferably about 0.001 to about 20% (w/w), more preferably about 0.01xe2x80x94about 10% (w/w), and still more preferably about 0.05xe2x80x94about 5% (w/w).
In the above-described o/w method, microcapsules may be produced by a method in which the complex is dispersed in an organic solvent solution of a biodegradable polymer, i.e., the s/o/w method.
(ii) In-water Drying Method (w/o/w method)
In this method, a solution of a biodegradable polymer in an organic solvent is first prepared. The concentration of the biodegradable polymer in the organic solvent solution is normally about 0.01 to about 80% (w/w), preferably about 0.1 to about 70% (w/w), and more preferably about 1 to about 60%, depending on the molecular weight of the biodegradable polymer, kind of organic solvent and so on. An aqueous dispersion of the complex is used as the internal aqueous phase. The concentration of the complex in the aqueous dispersion is, for example, about 10 to about 90% (w/v). The above-described aqueous dispersion of the complex is emulsified and dispersed in the organic solvent solution of the biodegradable polymer to form a w/o emulsion by known methods of dispersion using a turbine type mechanical stirrer, homogenizer and so on. This operation is conducted in such a way as to bring the weight ratio of the internal aqueous phase and the biodegradable polymer up to about 1:2, preferably about 1:3. The ratio of the internal aqueous phase and the organic solvent solution of the biodegradable polymer is 1:1,000 to 1:1 (v/v), preferably 1:100 to 1:5 (v/v), and more preferably 1:50 to 1:5 (v/v).
The w/o emulsion thus prepared is then added to another aqueous phase to form a w/o/w emulsion, followed by evaporation of the solvent in the oil phase, to yield microcapsules. This operation is conducted in accordance with term (i) above.
The sustained-release preparation of the present invention is preferably used in the form of fine particles. This is because sustained-release preparation does not cause undue pain to the patient when administered via an injection needle for ordinary subcutaneous or intramuscular injection. The mean particle diameter of the sustained-release preparation, for example, is about 0.1 to about 300 xcexcm, preferably about 1 to about 150 xcexcm, and more preferably about 2 to about 100 xcexcm.
In the present specification, a sustained-release preparation in fine particle form is also referred to as a microcapsule.
As used herein the term xe2x80x9cmicrocapsulexe2x80x9d may be referred to as xe2x80x9cmicrospherexe2x80x9d.
The sustained-release preparation of the present invention can, for example, be administered as microcapsules as such, or in the form of various dosage forms of non-oral preparations (e.g., intramuscular, subcutaneous or visceral injections or indwellable preparations, nasal, rectal or uterine transmucosal preparations etc.) or oral preparations (e.g., capsules such as hard capsules, soft capsules etc., solid preparations such as granules and powders etc., liquid preparations such as suspensions etc.).
In the present invention, the sustained-release preparation is preferably used for injection. When the sustained-release preparation is a microcapsule, for instance, it can be prepared as an aqueous suspension by suspending microcapsules in water, along with a dispersing agent (e.g., surfactants such as Tween 80 and HCO-60, polysaccharides such as carboxymethyl cellulose, sodium alginate and sodium hyaluronate etc.), a preservative (e.g., methyl paraben, propyl paraben etc.), an isotonizing agent (e.g., sodium chloride, mannitol, sorbitol, glucose etc.), etc., to yield a sustained-release preparation for injection of practical use. Alternatively, the sustained-release preparation of the present invention is prepared as an oily suspension by dispersing microcapsules, along with a vegetable oil such as sesame oil or corn oil with or without a phospholipid such as lecithin, or a medium-chain fatty acid triglyceride (e.g., MIGLYOL 812), to yield a sustained-release preparation for injection of practical use.
When the sustained-release preparation is a microcapsule, for instance, its mean particle size is chosen over the range from about 0.1 to about 300 xcexcm as long as the requirements concerning degree of dispersion and needle passage are met, when it is to be used as an injectable suspension. Preferably, the particle size falls within the range from about 1 to about 150 xcexcm, more preferably about 2 to about 100 xcexcm.
The above-described microcapsule can be prepared as a sterile preparation, without limitation by the method in which the entire production process is sterile, the method in which gamma rays is used as sterilant, and the method in which an antiseptic is added.
With low toxicity, the sustained-release preparation of the present invention can be safely used in mammals (e.g., humans, bovines, swines, dogs, cats, mice, rats, rabbits etc.).
Indications for the sustained-release preparation of the present invention vary according to the physiologically active substance used. For example, the sustained-release preparation of the present invention is effective in the treatment or prevention of diabetes mellitus etc. when the physiologically active substance is insulin; renal cancer, hepatitis C etc. when the physiologically active substance is interferon alpha; anemia etc. when the physiologically active substance is erythropoietin; developmental failure when the physiologically active substance is growth hormone, and neutropenia etc. after anticancer chemotherapy when the physiologically active substance is granulocyte colony-stimulating factor. When the physiologically active substance is erythropoietin, the sustained-release preparation of the present invention is also effective in promoting hematopoiesis for autotransfusion.
Depending on the type and content of the physiologically active substance, duration of physiologically active substance release, target disease, subject animal and other factors, the dose of the sustained-release preparation may be set at levels such that the physiologically active substance exhibits its action. The dose per administration of the physiologically active substance is chosen as appropriate over the range from about 0.0001 to about 10 mg/kg body weight for each adult, when the preparation is a 1-week preparation. More preferably, the dose may be chosen as appropriate over the range about about 0.0005 to about 1 mg/kg body weight.
The dose per administration of the sustained-release preparation is preferably chosen as appropriate over the range from about 0.0005 to about 50 mg/kg body weight for each adult. More preferably, the dose is chosen as appropriate over the range from about 0.0025 to about 10 mg/kg body weight. Dosing frequency can be chosen as appropriate, e.g., once weekly, once every two weeks or once every four weeks, depending on type, content and dosage form of the physiologically active substance, duration of physiologically active substance release, subject disease, subject animal and other factors.
Although the preparation of the present invention may be stored at normal temperature or in a cold place, it is preferable to store it in a cold place. Normal temperature and a cold place as mentioned herein are as defined by the Pharmacopoeia of Japan, specifically, 15 to 25xc2x0 C. for normal temperatures and under 15xc2x0 C. for cold places.